Objective(s): Growing evidence demonstrates that L-arginine/NO/cGMP/KATP channel pathway has a modulatory role in pain perception. Previous studies have shown that thymoquinone exerts antinociceptive effects; however, the mechanisms underlying antinociception induced by thymoquinone have not been fully clarified. The aim of the present study was to evaluate the role of L-arginine/NO/cGMP/KATP channel pathway in the central and peripheral antinociceptive effect of thymoquinone in rats.Materials and Methods: Rats were pretreated intraplantarly (IPL) or intracerebroventricularly (ICV) with L-arginine (the NO precursor), l-NAME (an NO synthase inhibitor), SNAP (an NO donor), methylene blue (a guanylyl cyclase inhibitor), glibenclamide (the blocker of KATP channel), and tetraethylammonium (TEA, a Kv channel blocker) before the injection of thymoquinone. Results: Local ipsilateral (20 and 40 μg, IPL) but not contralateral and ICV (4 and 8 μg) administration of thymoquinone caused a dose-dependent and significant antinociception in both early and late phases of the formalin test. Pretreatment of rats with L-arginine (100 μg, IPL or ICV) and SNAP (200 μg, IPL or ICV) increased while l-NAME (100 μg, IPL or 1 μg, ICV) and methylene blue (400 μg, IPL or ICV) decreased the antinociceptive effects of thymoquinone in the formalin test. The administration of TEA (IPL or ICV) did not modify but glibenclamide (50 μg, IPL or ICV) significantly abolished the peripheral and central antinociceptive effects of thymoquinone in both phases of the formalin test. Conclusion:The results of the present study indicate that L-arginine/NO/cGMP/KATP channel pathway participates in the central and peripheral antinociceptive effect of thymoquinone.